Targetable vector particles

ABSTRACT

A vector particle (eg., a retroviral vector particle) containing a chimeric envelope includes a receptor binding region that binds to a receptor of a target cell. The receptor of the target cell is other than the amphotropic cell receptor. The receptor binding region may be a receptor binding region of a human virus. A portion of the envelope gene may be deleted and the deleted portion is replaced with another receptor binding region or ligand. Such vector particles are targetable to a desired target cell or tissue, and may be administered directly to the desired target cell or tissue as part of a gene therapy procedure, or administered directly into the patient.

This is a Divisional of application Ser. No. 08/484,126 filed Jun. 7,1995, now U.S. Pat. No. 5,985,655, which is a continuation ofapplication Ser. No. 08/326,347, filed Oct. 20, 1994, abandoned, whichis a continuation of application Ser. No. 07/973,307, filed Nov. 9,1992, abandoned.

This invention relates to “targetable” vector particles. Moreparticularly, this invention relates to vector particles which include areceptor binding region that binds to a receptor of a target cell of ahuman or non-human animal.

Vector particles are useful agents for introducing gene(s) or DNA (RNA)into a cell, such as a eukaryotic cell. The gene(s) is controlled by anappropriate promoter. Examples of vectors which may be employed togenerate vector particles include prokaryotic vectors, such as bacterialvectors; eukaryotic vectors, including fungal vectors such as yeastvectors; and viral vectors such as DNA virus vectors, RNA virus vectors,and retroviral vectors. Retroviral vectors which have been employed forgenerating vector particles for introducing genes or DNA (RNA) into acell include Moloney Murine Leukemia Virus, spleen necrosis virus, andvectors derived from retroviruses such as Rous Sarcoma Virus and HarveySarcoma Virus. The term “introducing” as used herein encompasses avariety of methods of transferring genes or DNA (RNA) into a cell, suchmethods including transformation, transduction, transfection, andinfection.

Vector particles have been used for introducing DNA (RNA) into cells forgene therapy purposes. In general, such a procedure involves obtainingcells from a patient and using a vector particle to introduce desiredDNA (RNA) into the cells and then providing the patient with theengineered cells for a therapeutic purpose. It would be desirable toprovide alternative procedures for gene therapy. Such an alternativeprocedure would involve genetically engineering cells in vivo. In such aprocedure, a vector particle which includes the desired DNA (RNA) wouldbe administered directly to the target cells of a patient in vivo.

It is therefore an object of the present invention to provide genetherapy by introduction of a vector particle, such as, for example, aretroviral vector particle, directly into a desired target cell of apatient.

In accordance with an aspect of the present invention, there is provideda retroviral vector particle which includes a receptor binding region orligand that binds to a receptor of a target cell. The receptor of thetarget cell is a receptor other than the amphotropic cell receptor.

Retroviruses have an envelope protein which contains a receptor bindingregion. Applicants have found that retroviruses can be made “targetable”to a specific type of cell if the receptor binding region of theretrovirus, which may be amphotropic, ecotropic, or xenotropic, amongother types, is modified such that the receptor binding region of theenvelope protein includes a receptor binding region which binds to areceptor of a target cell. For example, at least a portion of thereceptor binding region of the envelope protein of the retrovirus isdeleted and replaced with a receptor binding region or a ligand whichbinds to a receptor of a target cell. Thus, there is provided aretroviral vector wherein at least a portion of the DNA (RNA) whichencodes the receptor binding region of the envelope protein of theretrovirus has been deleted and replaced with DNA (RNA) encoding areceptor binding region or a ligand which binds to a receptor of atarget cell.

In one embodiment, the retrovirus is a murine leukemia virus.

The envelope of murine leukemia viruses includes a protein known asgp70. Such viruses can be made “targetable” to a specific type of cellif a portion of the gp70 protein is deleted and replaced with a receptorbinding region or a ligand which binds to a receptor of a target cell.Thus, in a preferred embodiment, there is provided a retroviral vectorwherein a portion, but not all, of the DNA (RNA) encoding gp70 proteinhas been deleted and replaced with DNA (RNA) encoding a receptor bindingregion or a ligand which binds to a receptor of a target cell.

In general, gp70 protein includes the following regions: (i) thesecretory signal or “leader” sequence; (ii) the receptor binding domain;(iii) the hinge region; and (iv) the body portion. Preferably, at leasta portion of the DNA (RNA) encoding the receptor binding domain of gp70protein is deleted and replaced with DNA (RNA) encoding a receptorbinding region or a ligand which binds to a receptor of a target cell.More preferably, DNA (RNA) encoding the entire receptor binding domainof gp70 protein is deleted and replaced with DNA (RNA) encoding areceptor binding region or a ligand which binds to a receptor of atarget cell. In another embodiment, DNA. (RNA) encoding the entirereceptor binding domain of gp70 protein, plus all or a portion of theDNA (RNA) encoding the hinge region of gp70 protein is deleted andreplaced with DNA (RNA) encoding a receptor binding region or a ligandof a target cell.

The gp70 protein may be derived from an ecotropic murine leukemia virus,a xenotropic murine leukemia virus, or an amphotropic murine leukemiavirus. Ecotropic gp70 (or eco gp70) (SEQ ID NO:1) is a protein having469 amino acids, and is encoded by (SEQ ID:2). Amino acid residues 1-33constitute the leader sequence; amino acid residues 34-263 constitutethe receptor binding domain; amino acid residues 264-312 constitute thehinge region; and amino acid residues 313-469 constitute the bodyportion. Preferably, DNA (RNA) encoding at least a portion of thereceptor binding region is removed and replaced with DNA (RNA) encodinga receptor binding region or a ligand which binds to a receptor of atarget cell. More preferably, DNA (RNA) encoding some or all of aminoacid residues 34 to 263 (i.e., the receptor binding domain) is removedand replaced with DNA (RNA) encoding a receptor binding region or aligand which binds to a receptor of a target cell.

Xenotropic gp70 (or xeno gp70) (SEQ ID NO:3) has 443 amino acid residuesand is encoded by (SEQ ID NO:4). Amino acid residues 1-30 constitute theleader sequence; amino acid residues 31-232 constitute the receptorbinding domain; amino acid residues 233-286 constitute the hinge region;and amino acid residues 287-443 constitute the body portion. Preferably,DNA (RNA) encoding at least a portion of the receptor binding region isremoved and replaced with DNA (RNA) encoding a receptor binding regionor a ligand which binds to a receptor of a target cell. More preferably,DNA (RNA) encoding some or all of amino acid residues 31 to 232 isremoved and replaced with DNA (RNA) encoding a receptor binding regionor a ligand which binds to a receptor of a target cell.

Target cells to which the retroviral vector particle may bind include,but are not limited to, liver cells, T-cells, lymphocytes, endothelialcells, T4 helper cells, and macrophages. In one embodiment, theretroviral vector particle binds to a liver cell, and in particular tohepatocytes. To enable such binding, the retroviral vector particlecontains a chimeric protein encoded by DNA (RNA) in which at least aportion of the DNA (RNA) encoding the receptor binding domain of gp70protein is removed and is replaced with DNA (RNA) which encodes aprotein which binds to an asialoglycoprotein receptor (or ASG-R) ofhepatocytes.

Proteins which bind to the asialoglycoprotein receptor of liver cellsinclude, but are not limited to, asialoglycoproteins such as, forexample, alpha-1-acid glycoprotein (AGP), also known as orosomucoid, andasialofetuin. AGP is a natural high-affinity ligand for ASG-R. Theasialoglycoprotein receptor, or ASG-R, is expressed only by hepatocytes.The receptor is present at about 3×10⁵ copies per cell, and suchreceptors have a high affinity for asialoglycoproteins such as AGP.Thus, the engineering of retroviral vector particles to containasialoglycoprotein in place of the natural receptor binding domain ofgp70 generates retroviral vector particles which bind to theasialoglycoprotein receptor of hepatocytes, which provides for anefficient means of transferring genes of interest to liver cells.

Cell lines which generate retroviral vector particles that are capableof targeting the hepatocyte's asialoglycoprotein receptor without theremoval of the particle's terminal sialic acid groups by neuraminidasetreatment, can be developed by selection with the cytotoxic lectin,wheat germ agglutinin (WGA). Cell lines which express the retroviralproteins gag and pol become retroviral vector packaging cell lines afterthey are transfected with the plasmids encoding chimeric envelope genes.These cell lines express the corresponding chimeric gp 70 glycoproteins.Upon exposure to successively higher concentrations of WGA, theoutgrowth of cells which synthesize glycoproteins that lack terminalsialic acid groups, is favored. (Stanley, et al., Somatic Cell Genetics,Vol. 3, pgs. 391-405 (1977)). This selection permits the isolation ofcells which synthesize oligosaccharides terminating in galactosyl sugargroups. Such cells will allow the construction of packaging cell linesthat are capable of generating retroviral vector particles which targetthe asialoglycoprotein receptor. It is also possible to selectsubpopulations of packaging cells which have other distinct glycotypes,such cells yielding viral vectors that potentially are capable oftargeting cells other than hepatocytes. Macrophages, for example,express unique, high-mannose receptors. The PHA-resistant subpopulationwill have N-linked oligosaccharides which terminate in high-mannosegroups (Stanley, et al., In Vitro, Vol. 12, pgs. 208-215 (1976)).Therefore, such a cell population will be capable of producing viralvector particles capable of targeting macrophoges via this receptor.Cells with mutant glycotypes which synthesize other noveloligosaccharides after selection with other cytotoxic lectins may alsoprove to be useful in targeting vector particles to other cell typessuch as lymphocytes or endothelial cells.

In another embodiment, the receptor binding region is a receptor bindingregion of a human virus. In one embodiment, the receptor binding regionof a human virus is a hepatitis B virus surface protein binding region,and the target cell is a liver cell.

In another embodiment, the receptor binding region of a human virus isthe gp46 protein of HTLV-I virus, and the target cell is a T-cell.

In yet another embodiment, the receptor binding region of a human virusis the HIV gp120 CD4 binding region, and the target cell is a T4 helpercell.

In one embodiment, the retroviral vector may be of the LN series ofvectors, as described in Bender, et al., J. Virol., Vol. 61, pgs.1639-1649 (1987), and Miller, et al., Biotechniques, Vol. 7, pgs. 98-990(1989).

In another embodiment, the retroviral vector includes a multiplerestriction enzyme site, or multiple cloning site. The multiple cloningsite includes at least four cloning, or restriction enzyme sites,wherein at least two of the sites have an average frequency ofappearance in eukaryotic genes of less than once in 10,000 base pairs;i.e., the restriction product has an average size of at least 10,000base pairs.

In general, such restriction sites, also sometimes hereinafter referredto as “rare” sites, which have an average frequency of appearance ineukaryotic genes of less than once in 10,000 base pairs, contain a CGdoublet within their recognition sequence, such doublet appearingparticularly infrequently in the mammalian genome. Another measure ofrarity or scarcity of a restriction enzyme site in mammals is itsrepresentation in mammalian viruses, such as SV40. In general, an enzymewhose recognition sequence is absent in SV40 may be a candidate forbeing a “rare” mammalian cutter.

Examples of restriction enzyme sites having an average frequency ofappearance in eukaryotic genes of less than once in 10,000 base pairsinclude, but are not limited to the NotI, SnaBI, SalI, XhoI, ClaI, SacI,EagI, and SmaI sites. Preferred cloning sites are selected from thegroup consisting of NotI, SnaBI, SalI, and XhoI.

Preferably, the multiple cloning site has a length no greater than about70 base pairs, and preferably no greater than about 60 base pairs. Ingeneral, the multiple restriction enzyme site, or multiple cloning siteis located between the 5′ LTR and 3′ LTR of the retroviral vector. The5′ end of the multiple cloning site is no greater than about 895 basepairs from the 3′ end of the 5′ LTR, preferably at least about 375 basepairs from the 3′ end of the 5′ LTR. The 3′ end of the multiple cloningsite is no greater than about 40 base pairs from the 5′ end of the 3′LTR, and preferably at least 11 base pairs from the 5′ end of the 3′LTR.

Such vectors may be engineered from existing retroviral vectors throughgenetic engineering techniques known in the art such that the retroviralvector includes at least four cloning sites wherein at least two of thecloning sites are selected from the group consisting of the NotI, SnaBI,SalI, and XhoI cloning sites. In a preferred embodiment, the retroviralvector includes each of the NotI, SnaBI, SalI, and XhoI cloning sites.

Such a retroviral vector may serve as part of a cloning system for thetransfer of genes to such retroviral vector. Thus, there may be provideda cloning system for the manipulation of genes in a retroviral vectorwhich includes a retroviral vector including a multiple cloning site ofthe type hereinabove described, and a shuttle cloning vector whichincludes at least two cloning sites which are compatible with at leasttwo cloning sites selected from the group consisting of NotI, SnaBI,SalI, and XhoI located on the retroviral vector. The shuttle cloningvector also includes at least one desired gene which is capable of beingtransferred from said shuttle cloning vector to said retroviral vector.

The shuttle cloning vector may be constructed from a basic “backbone”vector or fragment to which are ligated one or more linkers whichinclude cloning or restriction enzyme recognition sites. Included in thecloning sites are the compatible, or complementary cloning siteshereinabove described. Genes and/or promoters having ends correspondingto the restriction sites of the shuttle vector may be ligated into theshuttle vector through techniques known in the art.

The shuttle cloning vector may be employed to amplify DNA sequences inprokaryotic systems. The shuttle cloning vector may be prepared fromplasmids generally used in prokaryotic systems and in particular inbacteria. Thus, for example, the shuttle cloning vector may be derivedfrom plasmids such as pBR322; pUC18; etc.

Such retroviral vectors are transfected or transduced into a packagingcell line, whereby there are generated infectious vector particles whichinclude the retroviral vector. In general, the vector is transfectedinto the packaging cell line along with a packaging defective helpervirus which includes genes encoding the gag and pol, and the envproteins of the virus. Representative examples of packaging cell linesinclude, but are not limited to, the PE501 and PA317 cell linesdisclosed in Miller, et al., Biotechniques, Vol. 7 pgs. 980-990 (1989).

The vector particles generated from the packaging cell line, which arealso engineered with a protein containing a receptor binding region thatbinds to a receptor of a target cell, said receptor being other than theamphotropic cell receptor, are targetable, whereby the receptor bindingregion enables the vector particles to bind to a target cell. Theretroviral vector particles thus may be directly administered to adesired target cell ex vivo, and such cells may then be administered toa patient as part of a gene therapy procedure.

Although the vector particles may be. administered directly to a targetcell, the vector particles may be engineered such that the vectorparticles are “injectable” as well as targetable; i.e., the vectorparticles are resistant to inactivation by human serum, and thus thetargetable vector particles may be administered to a patient byintravenous injection, and travel directly to a desired target cell ortissue without being inactivated by human serum.

The envelope of retroviruses also includes a protein known as p15E, andApplicants have found that retroviruses are susceptible to inactivationby human serum a a result of the action of complement protein(s) presentin serum on the p15E protein portion of the retrovirus. Applicants havefurther found that such retroviruses can be made resistant toinactivation by human serum by mutating such p15E protein.

In one embodiment, therefore, the retroviral vector is engineered suchthat a portion of the DNA (RNA) encoding p15E protein (shown in theaccompanying sequence listing as SEQ ID NO:7), has been mutated torender the vector particle resistant to inactivation by human serum;i.e., at least one amino acid but not all of the amino acids of the p15Eprotein has been changed, or mutated.

p15E protein is a viral protein having 196 amino acid residues. Inviruses, sometimes all 196 amino acid residues are present, and in otherviruses, amino acid residues 181 to 196 (known as the “r” peptide), arenot present, and the resulting protein is the “mature” form of p15Eknown as p12E. Thus, viruses can contain both the p15E and p12Eproteins. p15E protein is anchored in the viral membrane such that aminoacid residues residues 1 to 134 are present on the outside of the virus.Although this embodiment of the present invention is not to be limitedto any of the following reasoning, Applicants believe complementproteins may bind to this region whereby such binding leads toinactivation and/or lysis of the retrovirus. In particular, the p15Eprotein includes two regions, amino acid residues 39 to 61 (sometimeshereinafter referred to as region 1), and amino acid residues 101 to 123(sometimes hereinafter referred to as region 2), which Applicantsbelieve have an external location in the three-dimensional structure ofthe p15E protein; i.e., such regions are directly exposed to humanserum. Region 2 is a highly conserved region in many retroviruses, eventhough the amino acid sequences of this region are not identical in allretroviruses. Such regions are complement binding regions. Examples ofcomplement proteins which may bind to the complement binding regions areClS and ClQ, which bind to regions 1 and 2.

In order to inactivate the retrovirus, complement proteins bind to bothregion 1 and region 2. Thus, in a preferred embodiment, at least oneportion of DNA encoding a complement binding region of p15E protein hasbeen mutated. Such a mutation results in a change of at least one aminoacid residue of a complement binding region of p15E protein. The changein at least one amino acid residue of a complement binding region ofp15E protein prevents binding of a complement protein to the complementbinding region, thereby preventing complement inactivation of theretrovirus. In one embodiment, at least one amino acid residue in bothcomplement binding regions of p15E protein is changed, whereas inanother embodiment, at least one amino acid residue in one of thecomplement binding regions is changed.

It is to be understood, however, that the entire DNA sequence encodingp15E protein cannot be mutated because such a change renders the vectorsunsuitable for in vivo use.

In one embodiment, the mutation of DNA (RNA) encoding p15E protein maybe effected by deleting a portion of the p15E gene, and replacing thedeleted portion of the p15E gene, with fragment(s) or portion(s) of agene encoding another viral protein. In one embodiment, one portion ofDNA encoding the p15E protein is replaced with a fragment of the geneencoding the p21 protein, which is an HTLV-I transmembrane protein.HTLV-I virus has been found to be resistant to binding by complementproteins and thus HTLV-I is resistant to inactivation by human serum(Hoshino, et al., Nature, Vol. 310, pgs. 324-325 (1984)). Thus, in oneembodiment, there is also provided a retroviral vector particle whereina portion of the p15E protein has been deleted and replaced with aportion of another viral protein, such as a portion of the p21 protein.

p21 protein (as shown in the accompanying sequence listing as SEQ IDNO:8) is a protein having 176 amino acid residues, and which, inrelation to p15E, has significant amino acid sequence homology. In oneembodiment, at least amino acid residues 39 to 61, and 101 to 123 aredeleted from p15E protein, and replaced with amino acid residues 34 to56 and 96 to 118 of p21 protein. In one alternative, at least amino acidresidues 39 to 123 of p15E protein are deleted and replaced with aminoacid residues 34 to 118 of p21 protein.

In another embodiment, amino acid residues 39 to 69 of p15E protein aredeleted and replaced with amino acid residues 34 to 64 of p21 protein,and amino acid residues 96 to 123 of p15E protein are deleted andreplaced with amino acid residues 91 to 118 of p21 protein.

Vector particles generated from such packaging lines, therefore, are“targetable” and “injectable,” whereby such vector particles, uponadministration to a patient, travel directly to a desired target cell ortissue.

The targetable vector particles are useful for the introduction ofdesired heterologous genes into target cells ex vivo. Such cells maythen be administered to a patient as a gene therapy procedure, whereasvector particles which are targetable and injectable may be administeredin vivo to the patient, whereby the vector particles travel directly toa desired target cell.

Thus, preferably, the vectors or vector particles of the presentinvention further include at least one heterologous gene. Heterologousor foreign genes which may be placed into the vector or vector particlesinclude, but are not limited to, genes which encode cytokines orcellular growth factors, such as lymphokines, which are growth factorsfor lymphocytes. Other examples of foreign genes include, but are notlimited to, genes encoding Factor VIII, Factor IX, tumor necrosisfactors (TNF's), ADA, ApoE, ApoC, and Protein C.

The vectors of the present invention include one or more promoters.Suitable promoters which may be employed include, but are not limitedto, the retroviral LTR; the SV40 promoter; and the human cytomegalovirus(CMV) promoter described in Miller, et al., Biotechniques, Vol. 7, No.9, pgs 980-990 (1989), or any other promoter (e.g., cellular promoterssuch as eukaryotic cellular promoters including, but not limited to, thehistone, pol III, and B-actin promoters). Other viral promoters whichmay be employed include, but are not limited to, adenovirus promoters,TK promoters, and B19 parvovirus promoters. The selection of a suitablepromoter will be apparent to those skilled in the art from the teachingscontained herein.

The vectors of the present invention may contain regulatory elements,where necessary to ensure tissue specific expression of the desiredheterologous gene(s), and/or to regulate expression of the heterologousgene(s) in response to cellular or metabolic signals.

Although the invention has been described with respect to retroviralvector particles, other viral vector particles (such as, for example,adenovirus, adeno-associated virus, and Herpes Simplex virus particles),or synthetic particles may be constructed such that the vector particlesinclude a receptor binding region that binds to a receptor of a targetcell, wherein the receptor of a human target cell is other than theamphotropic cell receptor. Such vector particles are suitable for invivo administration to a desired target cell.

Advantages of the present invention include the ability to providevector particles which may be administered directly to a desired targetcell or tissues, whereby desired genes are delivered to the target cellor tissue, whereby the target cell or tissue may produce the proteinsexpressed by such genes.

This invention will now be described with respect to the followingexamples; however, the scope of the present invention is not intended tobe limited thereby.

BRIEF DESCRIPTION OF THE DRAWINGS

FIG. 1 is a map of plasmid pCee;

FIG. 2 is a map of plasmid pAGP-1;

FIG. 3 is a map of plasmid pAGP-3;

FIG. 4 is a map of plasmid pUC18RSVXeno;

FIG. 5 is a map of plasmid pAX2; and

FIG. 6 is a map of plasmid pAX6.

EXAMPLE 1

Plasmid pCee (FIG. 1), which contains the ecotropic murine leukemiavirus gp70 and p15E genes under the control of a CMV promoter, was cutwith AccI, and an AccI fragment encoding amino acid residues 1-312 ofthe eco gp70 protein was removed. Cloned into the AccI site was a PCRfragment containing the eco gp70 secretion signal (or leader, whichincludes amino acid residues 1-33 of eco gp70), followed by maturerabbit alpha-1 acid glycoprotein (amino acid residues 19-201) (Ray, etal., Biochemical and Biophysical Research Communications, Vol. 178, No.2, pgs. 507-513 (1991)). The amino acid sequence of rabbit alpha-1 acidglycoprotein is shown in (SEQ ID NO:5), and the DNA sequence encodingtherefor is shown in (SEQ ID NO:6). The resulting plasmid pAGP-1 (FIG.2) contains the eco gp70 leader sequence (amino acid residues 1-33 ofeco gp70), a sequence encoding the mature rabbit alpha-1 acidglycoprotein (amino acid residues 19-201), and a sequence encoding aminoacid residues 313 to 469 of eco gp70.

EXAMPLE 2

Plasmid pCee was cut with SalI and Pf1MI, and a SalI-Pf1MI fragmentencoding amino acid residues 1-262 of eco gp70 was removed. Cloned intothis site was a PCR generated SalI-Pf1MI fragment containing the ecogp70 leader sequence and the sequence encoding mature rabbit alpha-1acid glycoprotein. The resulting plasmid, pAGP-3 (FIG. 3) thus includesa sequence encoding the leader sequence of eco gp70, a sequence encodingmature rabbit alpha-1 acid glycoprotein; and a sequence encoding aminoacid residues 263 to 469 of eco gp70.

EXAMPLE 3

Plasmid pUC18RSVXeno (FIG. 4), which contains the xenotrophic murineleukemia virus gp70 and p15E genes under the control of an RSV promoter,was cut with AccI and StuI, and an AccI-StuI fragment encoding aminoacid residues 1-258 of xeno gp70 was removed. Cloned into this site wasa PCR generated AccI-StuI fragment encoding the xeno gp70 leader (aminoacid residues 1-30), and the mature rabbit alpha-1 acid glycoprotein.The resulting plasmid, pAX2 (FIG. 5), thus contains a sequence encodingthe xeno gp70 leader, a sequence encoding the mature rabbit alpha-1 acidglycoprotein, and amino acid residues 259-443 of xeno gp70.

EXAMPLE 4

Plasmid pUC18RSVXeno was cut with AccI and ClaI, and a fragment encodingamino acid residues 1-210 of xeno gp70 was removed. Cloned into thissite was a PCR generated AccI-Clal fragment encoding the xeno gp70leader, followed by mature rabbit alpha-1 acid glycoprotein. Theresulting plasmid, pAX6 (FIG. 6), thus includes a sequence encoding thexeno gp70 leader, a sequence encoding mature rabbit alpha-1 acidglycoprotein, and amino acid residues 211-443 of xeno gp70.

EXAMPLE 5

5×10⁵ GPL cells on 10 cm tissue culture plates were transfected (usingCaPO₄) with 30 μg/plate of one of plasmids pAGP-1, pAGP-3, pAX2, orpAX6. The CaPO₄ is removed 24 hours later and 10 ml of fresh D10 mediumis added for another 24 hours. The D10 medium is then removed andreplaced with serum free DX medium for another 24 hours. The DX mediumis then collected, filtered, and stored on ice. This supernatantcontains the vector particles.

The supernatants were then filtered and collected by standard proceduresand then centrifuged. After centrifugation, the virus pellets werereconstituted in a buffer containing 0.1M sodium acetate, 0.15M sodiumchloride, and 2 mM calcium chloride; the buffer was sterilized using aFalcon 0.2 millimicron tissue culture filter.

2.2 ml of concentrated supernatant containing viral particles generatedfrom pAGP-1 or pAGP-3, said viral particles sometimes hereinafterreferred to as Chimeric-1 or Chimeric-3, were loaded onto two disposableplastic columns which were alcohol sterilized and dried. To each column(1 cm×6 cm), one unit of neuraminidase from Clostridium perfringenswhich was bound to beaded agarose was added as a 2 ml suspension. Thisrepresents 1 ml of packed gel or unit of enzyme per column (15.7 mg ofagarose/ml and 28 units per gram of agarose). A unit is defined as theamount of neuraminidase which will liberate 1.0 micromole ofN-acetylneuraminic acid per minute from NAN-lactose at pH 5.0 and 37° C.

The columns were then washed with a large excess (50 ml) of the bufferhereinabove described to free the resin of all traces of freeneuraminidase and to sterilize the resin prior to incubation with virus.The columns were then dried, and the bottoms were sealed with caps andsecured with parafilm. The concentrated virus which was reconstituted inthe buffer (2.0 ml per sample) was then added to the resin. The topswere placed on the columns and secured with parafilm. The resin wasgently re-suspended by hand. The virus was then incubated with the resinfor 1 hour at room temperature with gentle rotation on a wheel. Thecolumns were checked periodically to ensure good mixing of resin andvirus.

At the end of the incubation period, the Chimera-1 and Chimera-3 viruseswere recovered by gentle vacuum filtration and collected into separatesterile 12×75 mm plastic polypropylene Falcon 2063 tubes. Recovery wasgreater than 90%, giving about 1.8 ml of desialated virus.

6-well plates containing about 105 receptor-positive (Hep G2) orreceptor-negative (SK HepI) human hepatocytes in 2 ml D10 media wereemployed as target cells. 24 hours after the cells were plated, 1 ml ofD10 was removed from the first well and 2 ml of neuraminidase-treated(or untreated as a control) viral supernatant containing Chimeric-1 orChimeric-3 was added and mixed well. 200 ul from the first well wasdiluted into the 2 ml present in the second well, was then mixed; andthen 200 ul from the second well was diluted into the 1.8 ml present inthe third well, thereby giving approximate dilutions of 2/3, 1/15, and1/150. 8 ug/ml of Polybrene was included in each well during thetransduction. The viral particles were left in contact with the cellsovernight, followed by removal of media containing viral particles, andreplaced with D10 containing 1,000 mg/ml of G418. The medium was changedwith fresh D10 and G418 every 4 to 5 days as necessary. G418-resistantcolonies were scored after 2 to 3 weeks.

EXAMPLE 6

The pre-packaging cell line GP8, which expresses the retroviral proteinsgag and pol, and the packaging cell lines derived from them which alsoexpress the chimeric gp70 glycoproteins encoded by the plasmids pAGP-1,pAGP-3, pAX2, or pAX6 were maintained in cell culture and exposed tosuccessively higher concentrations of wheat germ agglutinin; startingwith 15 ug/ml. The cell lines were maintained under WGA selection incell culture for 6 to 8 weeks until populations resistant to 40-50 ug/mlWGA were obtained. The latter were then subjected tofluoresence-activated cell sorting using FITC-conjugated lectins toenrich for the cells expressing the desired mutant glycotype (e.g.,FITC-Erythrina Cristagalli agglutinin for beta-D-galactosyl groups, andFITC-concanavalin A for alpha-D-mannosyl groups). Retroviral vectorpackaging and producer cell lines were then generated from the resultingpopulations by standard techniques.

It is to be understood, however, that the scope of the present inventionis not to be limited to the specific embodiments described above. Theinvention may be practiced other than as particularly described andstill be within the scope of the accompanying claims.

SEQUENCE LISTING (1) GENERAL INFORMATION: (iii) NUMBER OF SEQUENCES: 8(2) INFORMATION FOR SEQ ID NO: 1: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 469 amino acids (B) TYPE: amino acid (C) STRANDEDNESS: <Unknown>(D) TOPOLOGY: linear (ii) MOLECULE TYPE: protein (ix) FEATURE: (A)NAME/KEY: Ecotropic gp70 Protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO:1: Met Ala Arg Ser Thr Leu Ser Lys Pro Leu 5 10 Lys Asn Lys Val Asn ProArg Gly Pro Leu 15 20 Ile Pro Leu Ile Leu Leu Met Leu Arg Gly 25 30 ValSer Thr Ala Ser Pro Gly Ser Ser Pro 35 40 His Gly Val Tyr Asn Ile ThrTrp Glu Val 45 50 Thr Asn Gly Asp Arg Glu Thr Val Trp Ala 55 60 Thr SerGly Asn His Pro Leu Trp Thr Trp 65 70 Trp Pro Asp Leu Thr Pro Asp LeuCys Met 75 80 Leu Ala His His Gly Pro Ser Tyr Trp Gly 85 90 Leu Glu TyrGln Ser Pro Phe Ser Ser Pro 95 100 Pro Gly Pro Pro Cys Cys Ser Gly GlySer 105 110 Ser Pro Gly Cys Ser Arg Asp Cys Glu Glu 115 120 Pro Leu ThrSer Leu Thr Pro Arg Cys Asn 125 130 Thr Ala Trp Asn Arg Leu Lys Leu AspGln 135 140 Thr Thr His Lys Ser Asn Glu Gly Phe Tyr 145 150 Val Cys ProGly Pro His Arg Pro Arg Glu 155 160 Ser Lys Ser Cys Gly Gly Pro Asp SerPhe 165 170 Tyr Cys Ala Tyr Trp Gly Cys Glu Thr Thr 175 180 Gly Arg AlaTyr Trp Lys Pro Ser Ser Ser 185 190 Trp Asp Phe Ile Thr Val Asn Asn AsnLeu 195 200 Thr Ser Asp Gln Ala Val Gln Val Cys Lys 205 210 Asp Asn LysTrp Cys Asn Pro Leu Val Ile 215 220 Arg Phe Thr Asp Ala Gly Arg Arg ValThr 225 230 Ser Trp Thr Thr Gly His Tyr Trp Gly Leu 235 240 Arg Leu TyrVal Ser Gly Gln Asp Pro Gly 245 250 Leu Thr Phe Gly Ile Arg Leu Arg TyrGln 255 260 Asn Leu Gly Pro Arg Val Pro Ile Gly Pro 265 270 Asn Pro ValLeu Ala Asp Gln Gln Pro Leu 275 280 Ser Lys Pro Lys Pro Val Lys Ser ProSer 285 290 Val Thr Lys Pro Pro Ser Gly Thr Pro Leu 295 300 Ser Pro ThrGln Leu Pro Pro Ala Gly Thr 305 310 Glu Asn Arg Leu Leu Asn Leu Val AspGly 315 320 Ala Tyr Gln Ala Leu Asn Leu Thr Ser Pro 325 330 Asp Lys ThrGln Glu Cys Trp Leu Cys Leu 335 340 Val Ala Gly Pro Pro Tyr Tyr Glu GlyVal 345 350 Ala Val Leu Gly Thr Tyr Ser Asn His Thr 355 360 Ser Ala ProAla Asn Cys Ser Val Ala Ser 365 370 Gln His Lys Leu Thr Leu Ser Glu ValThr 375 380 Gly Gln Gly Leu Cys Ile Gly Ala Val Pro 385 390 Lys Thr HisGln Ala Leu Cys Asn Thr Thr 395 400 Gln Thr Ser Ser Arg Gly Ser Tyr TyrLeu 405 410 Val Ala Pro Thr Gly Thr Met Trp Ala Cys 415 420 Ser Thr GlyLeu Thr Pro Cys Ile Ser Thr 425 430 Thr Ile Leu Asn Leu Thr Thr Asp TyrCys 435 440 Val Leu Val Glu Leu Trp Pro Arg Val Thr 445 450 Tyr His SerPro Ser Tyr Val Tyr Gly Leu 455 460 Phe Glu Arg Ser Asn Arg His Lys Arg465 (2) INFORMATION FOR SEQ ID NO: 2: (i) SEQUENCE CHARACTERISTICS: (A)LENGTH: 1446 bases (B) TYPE: nucleic acid (C) STRANDEDNESS: single (D)TOPOLOGY: linear (ii) MOLECULE TYPE: viral DNA (xi) SEQUENCEDESCRIPTION: SEQ ID NO: 2: GGCTGCCGAC CCCGGGGGTG GACCATCCTC TAGACTGACATGGCGCGTTA AACGCTCTCA 60 AAACCCCTTA AAAATAAGGT TAACCCGCGA GGCCCCCTAATCCCCTTAAT TCTTCTGATG 120 CTCAGAGGGG TCAGTACTGC TTCGCCCGGC TCCAGTCCTCATCAAGTCTA TAATATCACC 180 TGGGAGGTAA CCAATGGAGA TCGGGAGACG GTATGGGCAACTTCTGGCAA CCACCCTCTG 240 TGGACCTGGT GGCCTGACCT TACCCCAGAT TTATGTATGTTAGCCCACCA TGGACCATCT 300 TATTGGGGGC TAGAATATCA ATCCCCTTTT TCTTCTCCCCCGGGGCCCCC TTGTTGCTCA 360 GGGGGCAGCA GCCCAGGCTG TTCCAGAGAC TGCGAAGAACCTTTAACCTC CCTCACCCCT 420 CGGTGCAACA CTGCCTGGAA CAGACTCAAG CTAGACCAGACAACTCATAA ATCAAATGAG 480 GGATTTTATG TTTGCCCCGG GCCCCACCGC CCCCGAGAATCCAAGTCATG TGGGGGTCCA 540 GACTCCTTCT ACTGTGCCTA TTGGGGCTGT GAGACAACCGGTAGAGCTTA CTGGAAGCCC 600 TCCTCATCAT GGGATTTCAT CACAGTAAAC AACAATCTCACCTCTGACCA GGCTGTCCAG 660 GTATGCAAAG ATAATAAGTG GTGCAACCCC TTAGTTATTCGGTTTACAGA CGCCGGGAGA 720 CGGGTTACTT CCTGGACCAC AGGACATTAC TGGGGCTTACGTTTGTATGT CTCCGGACAA 780 GATCCAGGGC TTACATTTGG GATCCGACTC AGATACCAAAATCTAGGACC CCGCGTCCCA 840 ATAGGGCCAA ACCCCGTTCT GGCAGACCAA CAGCCACTCTCCAAGCCCAA ACCTGTTAAG 900 TCGCCTTCAG TCACCAAACC ACCCAGTGGG ACTCCTCTCTCCCCTACCCA ACTTCCACCG 960 GCGGGAACGG AAAATAGGCT GCTAAACTTA GTAGACGGAGCCTACCAAGC CCTCAACCTC 1020 ACCAGTCCTG ACAAAACCCA AGAGTGCTGG TTGTGTCTAGTAGCGGGACC CCCCTACTAC 1080 GAAGGGGTTG CCGTCCTGGG TACCTACTCC AACCATACCTCTGCTCCAGC CAACTGCTCC 1140 GTGGCCTCCC AACACAAGTT GACCCTGTCC GAAGTGACCGGACAGGGACT CTGCATAGGA 1200 GCAGTTCCCA AAACACATCA GGCCCTATGT AATACCACCCAGACAAGCAG TCGAGGGTCC 1260 TATTATCTAG TTGCCCCTAC AGGTACCATG TGGGCTTGTAGTACCGGGCT TACTCCATGC 1320 ATCTCCACCA CCATACTGAA CCTTACCACT GATTATTGTGTTCTTGTCGA ACTCTGGCCA 1380 AGAGTCACCT ATCATTCCCC CAGCTATGTT TACGGCCTGTTTGAGAGATC CAACCGACAC 1440 AAAAGA 1446 (2) INFORMATION FOR SEQ ID NO: 3:(i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 453 amino acids (B) TYPE:amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY:xenotropic gp70protein (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 3: Met Glu Gly Ser Ala PheSer Lys Pro Leu 5 10 Lys Asp Lys Ile Asn Pro Trp Gly Pro Leu 15 20 IleVal Met Gly Ile Leu Val Arg Ala Gly 25 30 Ala Ser Val Gln Arg Asp SerPro His Gln 35 40 Ile Phe Asn Val Thr Trp Arg Val Thr Asn 45 50 Leu MetThr Gly Gln Thr Ala Asn Ala Thr 55 60 Ser Leu Leu Gly Thr Met Thr AspThr Phe 65 70 Pro Lys Leu Tyr Phe Asp Leu Cys Asp Leu 75 80 Pro Lys LeuTyr Phe Asp Leu Cys Asp Leu 85 90 Val Gly Asp Tyr Trp Asp Asp Pro GluPro 95 100 Asp Ile Gly Asp Gly Cys Arg Thr Pro Gly 105 110 Gly Arg ArgArg Thr Arg Leu Tyr Asp Phe 115 120 Tyr Val Cys Pro Gly His Thr Val ProIle 125 130 Gly Cys Gly Gly Pro Gly Glu Gly Tyr Cys 135 140 Gly Lys TrpGly Cys Glu Thr Thr Gly Gln 145 150 Ala Tyr Trp Lys Pro Ser Ser Ser TrpAsp 155 160 Leu Ile Ser Leu Lys Arg Gly Asn Thr Pro 165 170 Lys Asp GlnGly Pro Cys Tyr Asp Ser Ser 175 180 Val Ser Ser Gly Val Gln Gly Ala ThrPro 185 190 Gly Gly Arg Cys Asn Pro Leu Val Leu Glu 195 200 Phe Thr AspAla Gly Arg Lys Ala Ser Trp 205 210 Asp Ala Pro Lys Val Trp Gly Leu ArgLeu 215 220 Tyr Arg Ser Thr Gly Ala Asp Pro Val Thr 225 230 Arg Phe SerLeu Thr Arg Gln Val Leu Asn 235 240 Val Gly Pro Arg Val Pro Ile Gly ProAsn 245 250 Pro Val Ile Thr Asp Gln Leu Pro Pro Ser 255 260 Gln Pro ValGln Ile Met Leu Pro Arg Pro 265 270 Pro His Pro Pro Pro Ser Gly Thr ValSer 275 280 Met Val Pro Gly Ala Pro Pro Pro Ser Gln 285 290 Gln Pro GlyThr Gly Asp Arg Leu Leu Asn 295 300 Leu Val Glu Gly Ala Tyr Gln Ala LeuAsn 305 310 Leu Thr Ser Pro Asp Lys Thr Gln Glu Cys 315 320 Trp Leu CysLeu Val Ser Gly Pro Pro Tyr 325 330 Tyr Glu Gly Val Ala Val Leu Gly ThrTyr 335 340 Ser Asn His Thr Ser Ala Pro Ala Asn Cys 345 350 Ser Val AlaSer Gln His Lys Leu Thr Leu 355 360 Ser Glu Val Thr Gly Gln Gly Leu CysVal 365 370 Gly Ala Val Pro Lys Thr His Gln Ala Leu 375 380 Cys Asn ThrThr Gln Lys Thr Ser Asp Gly 385 390 Ser Tyr Tyr Leu Ala Ala Pro Ala GlyThr 395 400 Ile Trp Ala Cys Asn Thr Gly Leu Thr Pro 405 410 Cys Leu SerThr Thr Val Leu Asn Leu Thr 415 420 Thr Asp Tyr Cys Val Leu Val Glu LeuTrp 425 430 Pro Lys Val Thr Tyr His Ser Pro Asp Tyr 435 440 Val Tyr GlyGln Phe Glu Lys Lys Thr Lys 445 450 Tyr Lys Arg (2) INFORMATION FOR SEQID NO: 4: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 1356 bases (B) TYPE:nucleic acid (C) STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULETYPE: viral DNA (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 4: GCGACAACTCCTCCAGCCGG GAACAGCATG GAAGGTTCAG CGTTCTCAAA ACCCCTTAAA 60 GATAAGATTAACCCGTGGGG CCCCCTAATA GTTATGGGGA TCTTGGTGAG GGCAGGAGCT 120 TCGGTACAACGTGACAGCCC TCACCAGATC TTCAATGTTA CTTGGAGAGT TACCAACCTA 180 ATGACAGGACAAACAGCTAA CGCCACCTCC CTCCTGGGGA CGATGACAGA CACCTTCCCT 240 AAACTATATTTTGACCTGTG TGATTTAGTA GGAGACTACT GGGATGACCC AGAACCCGAT 300 ATTGGGGATGGTTGCCGCAC TCCCGGGGGA AGAAGAAGGA CAAGACTGTA TGACTTCTAT 360 GTTTGCCCCGGTCATACTGT ACCAATAGGG TGTGGAGGGC CGGGAGAGGG CTACTGTGGC 420 AAATGGGGATGTGAGACCAC TGGACAGGCA TACTGGAAGC CATCATCATC ATGGGACCTA 480 ATTTCCCTTAAGCGAGGAAA CACTCCTAAG GATCAGGGCC CCTGTTATGA TTCCTCGGTC 540 TCCAGTGGCGTCCAGGGTGC CACACCGGGG GGTCGATGCA ACCCCCTGGT CTTAGAATTC 600 ACTGACGCGGGTAGAAAGGC CAGCTGGGAT GCCCCCAAAG TTTGGGGACT AAGACTCTAT 660 CGATCCACAGGGGCCGACCC GGTGACCCGG TTCTCTTTGA CCCGCCAGGT CCTCAATGTA 720 GGACCCCGCGTCCCCATTGG GCCTAATCCC GTGATCACTG ACCAGCTACC CCCATCCCAA 780 CCCGTGCAGATCATGCTCCC CAGGCCTCCT CATCCTCCTC CTTCAGGCAC GGTCTCTATG 840 GTACCTGGGGCTCCCCCGCC TTCTCAACAA CCTGGGACGG GAGACAGGCT GCTAAATCTG 900 GTAGAAGGAGCCTACCAAGC ACTCAACCTC ACCAGTCCTG ACAAAACCCA AGAGTGCTGG 960 TTGTGTCTGGTATCGGGACC CCCCTACTAC GAAGGGCTTG CCGTCCTAGG TACCTACTCC 1020 AACCATACCTCTGCCCCAGC TAACTGCTCC GTGGCCTCCC AACACAAGCT GACCCTGTCC 1080 GAAGTAACCGGACAGGGACT CTGCGTAGGA GCAGTTCCCA AAACCCATCA GGCCCTGTGT 1140 AATACCACCCAGAAGACGAG CGACGGGTCC TACTATCTGG CTGCTCCCGC CGGGACCATC 1200 TGGGCTTGCAACACCGGGCT CACTCCCTGC CTATCTACTA CTGTACTCAA CCTCACCACC 1260 GATTACTGTGTCCTGGTTGA GCTCTGGCCA AAGGTAACCT ACCACTCCCC TGATTATGTT 1320 TATGGCCAGTTTGAAAAGAA AACTAAATAT AAAAGA 1356 (2) INFORMATION FOR SEQ ID NO: 5: (i)SEQUENCE CHARACTERISTICS: (A) LENGTH: 201 amino acids (B) TYPE: aminoacid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii) MOLECULETYPE: protein (ix) FEATURE: (A) NAME/KEY:rabbit alpha-1-acidglycoprotein (x) PUBLICATION INFORMATION (xi) SEQUENCE DESCRIPTION: SEQID NO: 5: Met Ala Leu Pro Trp Ala Leu Ala Val Leu 5 10 Ser Leu Leu ProLeu Leu His Ala Gln Asp 15 20 Pro Ala Cys Ala Asn Phe Ser Thr Ser Pro 2530 Ile Thr Asn Ala Thr Leu Asp Gln Leu Ser 35 40 His Lys Trp Phe Phe ThrAla Ser Ala Phe 45 50 Arg Asn Pro Lys Tyr Lys Gln Leu Val Gln 55 60 HisThr Gln Ala Ala Phe Phe Tyr Phe Thr 65 70 Ala Ile Lys Glu Glu Asp ThrLeu Leu Leu 75 80 Arg Glu Tyr Ile Thr Thr Asn Asn Thr Cys 85 90 Phe TyrAsn Ser Ser Ile Val Arg Val Gln 95 100 Arg Glu Asn Gly Thr Leu Ser LysHis Asp 105 110 Gly Ile Arg Asn Ser Val Ala Asp Leu Leu 115 120 Leu LeuArg Asp Pro Gly Ser Phe Leu Leu 125 130 Val Phe Phe Ala Gly Lys Glu GlnAsp Lys 135 140 Gly Met Ser Leu Tyr Thr Asp Lys Pro Lys 145 150 Ala SerThr Glu Gln Leu Glu Glu Phe Tyr 155 160 Glu Ala Leu Thr Cys Leu Gly MetAsn Lys 165 170 Thr Glu Val Val Tyr Thr Asp Trp Thr Lys 175 180 Asp LeuCys Glu Pro Leu Glu Lys Gln His 185 190 Glu Glu Glu Arg Lys Lys Glu LysAla Glu 195 200 Ser (2) INFORMATION FOR SEQ ID NO: 6 (i) SEQUENCECHARACTERISTICS: (A) LENGTH: 759 bases (B) TYPE: nucleic acid (C)STRANDEDNESS: single (D) TOPOLOGY: linear (ii) MOLECULE TYPE: genomicDNA (x) PUBLICATION INFORMATION: (A) AUTHORS: Ray, et al. (B) TITLE: (C)JOURNAL: Biochem. and Biophys. Res. Comm. (D) VOLUME: 178 (E) ISSUE: NO.2 (F) PAGES: 507-513 (G) DATE: 1991 (xi) SEQUENCE DESCRIPTION: SEQ IDNO: 6: AGCTCTGCCT GGCTCCAGCG CCTCTGTGTC TCAGCATGGC CCTGCCCTGG GCCCTCGCCG60 TCCTGAGCCT CCTCCCTCTG CTGCATGCCC AGGACCCAGC GTGTGCCAAC TTCTCGACCA 120GCCCTATCAC CAATGCCACC CTGGACCAGC TCTCCCACAA GTGGTTTTTT ACCGCCTCGG 180CCTTCCGGAA CCCCAAGTAC AAGCAGCTGG TGCAGCATAC CCAGGCGGCC TTTTTCTACT 240TCACCGCCAT CAAAGAGGAG GACACCTTGC TGCTCCGGGA GTACATAACC ACGAACAACA 300CGTGCTTCTA TAACTGCAGC ATCGTGAGGG TCCAGAGAGA GAATGGGACC CTCTCCAAAC 360ACGACGGCAT ACGAAATAGC GTGGCCGACC TGCTGCTCCT CAGGGACCCC GGGAGCTTCC 420TCCTCGTCTT CTTCGCTGGG AAGGAGCAGG ACAAGGGAAT GTCCTTCTAC ACCGACAAGC 480CCAAGGCCAG CCCGGAACAA CTGGAAGAGT TCTACGAAGC CCTCACGTGC CTGGGCATGA 540ACAAGACGGA AGTCGTCTAC ACTGACTGGA CAAAGGATCT GTGCGAGCCG CTGGAGAAGC 600AACACGAGGA GGAGAGGAAG AAGGAAAAGG CAGAGTCATA GGGCACAGCA CCGGCTCCGG 660GACTCGGGGC CCACCCCCTG CACCTGCCTT TTTGTTTGTT TTGTAAATCT CTGTTCTTTC 720CCATGGTTGC ATCAATAAAA CTGCTGGACC AGTAAAAAA 759 (2) INFORMATION FOR SEQID NO: 7: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 196 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY: ecotropic p15Eprotein. (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 7: Glu Pro Val Ser LeuThr Leu Ala Leu Leu 5 10 Leu Gly Gly Leu Thr Met Gly Gly Ile Ala 15 20Ala Gly Ile Gly Thr Gly Thr Thr Ala Leu 25 30 Met Ala Thr Gln Gln PheGln Gln Leu Gln 35 40 Ala Ala Val Gln Asp Asp Leu Arg Glu Val 45 50 GluLys Ser Ile Ser Asn Leu Glu Lys Ser 55 60 Leu Thr Ser Leu Ser Glu ValVal Leu Gln 65 70 Asn Arg Arg Gly Leu Asp Leu Leu Phe Leu 75 80 Lys GluGly Gly Leu Cys Ala Ala Leu Lys 85 90 Glu Glu Cys Cys Phe Tyr Ala AspHis Thr 95 100 Gly Leu Val Arg Asp Ser Met Ala Lys Leu 105 110 Arg GluArg Leu Asn Gln Arg Gln Lys Leu 115 120 Phe Glu Ser Thr Gln Gly Trp PheGlu Gly 125 130 Leu Phe Asn Arg Ser Pro Trp Phe Thr Thr 135 140 Leu IleSer Thr Ile Met Gly Pro Leu Ile 145 150 Val Leu Leu Met Ile Leu Leu PheGly Pro 155 160 Cys Ile Leu Asn Arg Leu Val Gln Phe Val 165 170 Lys AspArg Ile Ser Val Val Gln Ala Leu 175 180 Val Leu Thr Gln Gln Tyr His GlnLeu Lys 185 190 Pro Ile Glu Tyr Glu Pro 195 (2) INFORMATION FOR SEQ IDNO: 8: (i) SEQUENCE CHARACTERISTICS: (A) LENGTH: 176 amino acids (B)TYPE: amino acid (C) STRANDEDNESS: <Unknown> (D) TOPOLOGY: linear (ii)MOLECULE TYPE: protein (ix) FEATURE: (A) NAME/KEY: HTLV-I p21 protein(x) PUBLICATION INFORMATION: (A) AUTHORS: Malik, et al. (B) TITLE: (C)JOURNAL: J. Gen. Virol. (D) VOLUME: 69 (E) ISSUE: (F) PAGES: 1695-1710(G) DATE: 1988 (xi) SEQUENCE DESCRIPTION: SEQ ID NO: 8: Ala Val Pro ValAla Val Trp Leu Val Ser 5 10 Ala Leu Ala Met Gly Ala Gly Val Ala Gly 1520 Arg Ile Thr Gly Ser Met Ser Leu Ala Ser 25 30 Gly Lys Ser Leu Leu HisGlu Val Asp Lys 35 40 Asp Ile Ser Gln Leu Thr Gln Ala Ile Val 45 50 LysAsn His Lys Asn Leu Leu Lys Ile Ala 55 60 Gln Tyr Ala Ala Gln Asn ArgArg Gly Leu 65 70 Asp Leu Leu Phe Trp Glu Gln Gly Gly Leu 75 80 Cys LysAla Leu Gln Glu Gln Cys Cys Phe 85 90 Leu Asn Ile Thr Asn Ser His ValSer Ile 95 100 Leu Gln Glu Arg Pro Pro Leu Glu Asn Arg 105 110 Val LeuThr Gly Trp Gly Leu Asn Trp Asp 115 120 Leu Gly Leu Ser Gln Trp Ala ArgGlu Ala 125 130 Leu Gln Thr Gly Ile Thr Leu Val Ala Leu 135 140 Leu LeuLeu Val Ile Leu Ala Gly Pro Cys 145 150 Ile Leu Arg Gln Leu Arg His LeuPro Ser 155 160 Arg Val Arg Tyr Pro His Tyr Ser Leu Ile 165 170 Asn ProGlu Ser Ser Leu 175

What is claimed is:
 1. A method of introducing at least one heterologousgene into a target cell, comprising: administering to said target cell aretroviral vector particle, said retroviral vector particle including(i) a retroviral envelope protein, which includes a receptor bindingregion, a hinge region, and a body region, wherein a portion of saidretroviral envelope protein is deleted and a receptor binding region ora ligand that binds to a receptor of a target cell is inserted into saiddeleted portion, said receptor of a target cell being other than theamphotropic cell receptor, and wherein the only portion of theretroviral envelope protein that is deleted is (a) a portion or all ofthe receptor binding region, (b) a portion of the receptor bindingregion and a portion or all of the hinge region, or (c) all of thereceptor binding region and a portion or all of the hinge region, and(ii) at least one heterologous gene.
 2. The method of claim 1 whereinsaid vector particles are administered ex vivo.
 3. The method of claim 1wherein said vector particles are administered in vivo.
 4. A method ofintroducing at least one heterologous gene into a target cell,comprising: administering to said target cell a retroviral vectorparticle, said retroviral vector particle including (i) a retroviralenvelope protein, which includes a receptor binding region, a hingeregion, and a body region, wherein a portion of said retroviral envelopeprotein is deleted and a receptor binding region or a liquid that bindsto a receptor of a target cell is inserted into said deleted portion,said receptor of a target cell being other than the amphotropic cellreceptor, and wherein the only portion of the retroviral envelopeprotein is deleted is a portion or all of the receptor binding region,and (ii) at least one heterologous gene.
 5. The method of claim 4wherein said vector particles are administered ex vivo.
 6. The method ofclaim 4 wherein said vector particles are administered in vivo.
 7. Amethod of introducing at least one heterologous gene into a target cell,comprising: administering to said target cell a retroviral vectorparticle, said retroviral vector particle including (i) a retroviralenvelope protein, which includes a receptor binding region, a hingeregion, and a body region, wherein a portion of said retroviral envelopeprotein is deleted and a receptor binding region or a ligand that bindsto a receptor of a target cell is inserted into said deleted portion,said receptor of a target cell being other than the amphototropic cellreceptor, and wherein the only portion of the retroviral envelopeprotein that is deleted is a portion of the receptor binding region anda portion or all of the hinge region, and (ii) at least one heterologousgene.
 8. The method of claim 7 wherein said vector particles areadministered ex vivo.
 9. The method of claim 7 wherein said vectorparticles are administered in vivo.
 10. A method of introducing at leastone heterologous gene into a target cell, comprising: administering tosaid target cell a retroviral vector particle, said retroviral vectorparticle including (i) a retroviral envelope protein, which includes areceptor binding region, a hinge region, and a body region, wherein aportion of said retroviral envelope protein is deleted and a receptorbinding region or a ligand that binds to a receptor of a target cell isinserted into said deleted portion, said receptor of a target cell beingother than the amphotropic cell receptor, and wherein the only portionof the retroviral envelope protein that is deleted is a portion of thereceptor binding region and a portion or all of the hinge region, and(ii) at least one heterologous gene.
 11. The method of claim 10 whereinsaid vector particles are administered ex vivo.
 12. The method of claim10 wherein said vector particles are administered in vivo.